Chorioamnionitis (CAM), or inflammation of the fetal membranes, is often the result of ascending bacterial infection and is a major contributor to premature birth, neonatal sepsis, and long-term disability and morbidity in babies. Unfortunately, CAM is often asymptomatic and not easily diagnosed in time to prevent maternal and fetal adverse outcomes. Solving this problem is hamstrung by a limited understanding of early steps in disease pathogenesis. Despite its relatively simple structure, surprisingly little is known about how fetal membranes participate in immune defense against potential pathogens. Defining the cellular and molecular basis of innate immunity within the membranes promises to reveal actionable, host-based targets for diagnosis, prevention and therapy against CAM. Our objective is to define specific contributions of macrophages to fetal membrane immunology in the context of bacterial CAM caused by the common pathogen Group B Streptococcus (GBS). Macrophages in the gravid uterus balance host defense activities with pregnancy-specific actions such as promoting placental development and governing immune tolerance between mother and fetus. It is fascinating that both maternal (decidual) and fetal (placental) macrophages are present at the maternal-fetal interface, yet their specific roles in innate immunity are unknown. We hypothesize (1) that maternal and fetal macrophages make unique contributions both to host defense and tissue inflammatory responses during bacterial infection and (2) that the common CAM pathogen, GBS, evades innate immunity by resisting the oxidative stress within these macrophages. We will test this hypothesis through three Aims. In Aim 1 we will determine the extent to which maternal and fetal macrophages contribute to the natural history of CAM in vivo using a model of ascending GBS infection. In Aim 2 we will identify the extent to which GBS survival within macrophages depends upon the NADH peroxidase (npx) or other genotype-specific intracellular survival defenses. Mutagenesis studies will be conducted to better understand the impact of npx and other GBS mutations on survivability and disease in vivo, and to classify bacterial and host genes important for the process using RNA sequencing. Additional genotypes will be examined for variation in the ability to survive inside decidual and placental macrophages and persist in the presence of antibiotics commonly used to treat GBS infections. Lastly, in Aim 3 we will define the paracrine contribution of macrophages within the human fetal membrane during infection. For this aim, we will take a deconstructive approach, populating a microfluidic, instrumented fetal membrane-on-chip with decidual or placental macrophages, decidual stromal cells, trophoblasts and amnion epithelial cells. We will test the sub-hypothesis that distinct macrophage types contribute uniquely to inflammatory quiescence within uninfected membranes and amplify proinflammatory responses upon microbial threat in specific manners. These studies will shed new light on reproductive immunology and accelerate the development of new diagnostic, prognostic, and therapeutic interventions that support healthy pregnancies.